The three-dimensional structure of a cell-cell signaling peptide determines how it interacts with its cognate receptor and with various degradation enzymes. An enigmatic, poorly understood peptide modification-the enzymatic epimerization of a single amino acid residue-results in the formation of a D-amino acid-containing peptide (DAACP). Despite significant progress in DAACP characterization, discovery efforts are hampered by shortfalls in current technology. Why do we think there are unknown DAACPs? More than 30 have been reported in a surprising range of animals and organs including the brain; in mammals an undetermined enzyme activity converts several all-L-amino acid-containing peptides into DAACPs, and a unique aminopeptidase in nervous tissue is capable of degrading DAACPs. Accordingly, we hypothesize that DAACPs are widely present in nervous and endocrine tissues throughout the Metazoa. The overarching goal is to characterize the DAACP peptidome and determine the function of the newly uncovered DAACPs. We will create a comprehensive three-stage DAACP discovery funnel (Aim 1): (1) putative DAACP candidates are identified without the need for peptide standards; (2) the presence of the D-amino acid in the putative DAACP is confirmed; (3) using appropriate peptide standards and semi-purified peptides, the DAACPs are sequenced for absolute confirmation. Specifically, the funnel consists of linked analytical approaches: separation and characterization of isobaric peptides, analyses of peptides resistant to enzymatic digestion, separation/digestion of the peptides into their component amino acids for characterization via multiple reaction monitoring and chiral amino acid capillary electrophoresis, and finally, chiral tandem mass spectrometric peptide sequencing hyphenated to in silico structure determination. After funnel optimization, several model organisms will be used in the discovery phase: the exceptional neurophysiological model Aplysia californica with its known and putative DAACPs, and the regenerative planarian model with its ease of genetic manipulations (Aim 2); followed by experiments in mice and rat endocrine and selected nervous tissues (Aim 3). Most signaling peptide systems are ancient and well conserved; it appears that DAACPs are also. These comparative studies allow common biochemical pathways and physiologies to guide subsequent peptide bioactivity tests. Characterizing unknown DAACPs, mapping them to specific locations, correlating their levels to an animal's physiological state, and even electrophysiological testing combines to provide unparalleled information on their function (Aim 4). These research efforts are timely given the wealth of genomic and peptidome information available for our selected animal models and the recent discovery of enzymatic and biochemical data suggesting the presence of DAACPs. The discovery of new signaling DAACPs will improve our understanding of the functioning of the nervous and endocrine systems, and the discovery funnel will have application to a range of fundamental and applied investigations. PUBLIC HEALTH RELEVANCE: Peptides act as hormones, trophic factors, and transmitters; they are involved in many aspects of organism homeostasis, and so it is not surprising that their misregulation has profound effects on health. A suite of analytical approaches are being developed to characterize a rare posttranslational modification to peptides, the switch of a single amino acid from the L-form to the D-form. D-amino acid-containing peptides have been implicated as biomarkers of several diseases and this trend will continue as more of these novel cell-to-cell signaling peptides are characterized, their functions determined, and importantly, they reveal potential pharmaceutical targets.